Funds are requested to carry out projects on the function of proteins of the Crisp superfamily in the male reproductive tract. Three Crisp genes encode proteins in an organ-specific manner: Crisp-2 is testis- specific, Crisp-3 is submandibular gland-specific and Crisp-1 is expressed primarily in the epididymis. Crisp genes are found in mammals as diverse as rats, guinea pigs, mice and humans and so must have an important function in the organs within which they are expressed. Crisp-1 proteins are synthesized and secreted by the epididymal epithelium and subsequently bind to sperm in a domain-specific manner in rats. Protein D, which binds both loosely and tightly, localizes to the head of the sperm on the plasma membrane overlying the acrosome and has been shown to play a role in fertilization. Protein E binds specifically to plasma membrane of the tail, and also participates in fertilization. Proteins D and E have significant homology to helothermine (a salivary gland toxin that blocks ryanodine receptors) and to a number of insect and snake venom proteins that are capable of regulating ion channels. We hypothesize that proteins D and E function to regulate sperm ion fluxes, particularly calcium. In the first specific aim we explore the roles of Crisp-1 during sperm-egg fusion. In the second aim, we explore their role in capacitation and the acrosome reaction, both processes that require large ion fluxes. Finally, in the last specific aim we address the question of mechanism(s) that mediate binding of protein D and E to sperm in the epididymis. We hypothesize that the signal for domain localization resides in the NH2 terminus of the molecule and the carboxyl terminus, where 14 of the 16 cysteines are found, provides the plasma membrane binding domain. These studies will contribute significant knowledge on the role of a potentially important group of proteins both in the physiology of the epididymis and in fertilization.